The forces involved in ligand binding by antibodies and substrate binding by enzymes is similar, viz., hydrogen bonding, electrostatic interaction and hydrophobic effect. The energy obtained from enzyme-substrate binding may be visualized to force electronic strain in the substrate and facilitate the formation of a transition state. There is strong evidence for the theory that enzymes bind the transition state of the reaction they catalyze better than the ground state, resulting in a reduced free energy of activation for the reaction (1). The transition state theory is based upon the observation that the reactants for a chemical reaction normally exist at a ground state energy level. In order for a reaction to proceed, the energy level of the reactants must be raised to that required to form transition state intermediate(s). A successful catalyst may function to reduce the energy requirement for the formation of such transition state intermediate compound(s). This has come to be known as the transition state theory of enzymatic catalysis.
Other factors that may facilitate enzymatic catalysis include the proximity and orientation effects-apposition of correctly oriented reactants within the active site of the enzyme would reduce the requirement for a large number of random collisions prior to a productive reactant interaction. In principle, antibodies could catalyze chemical reactions by similar means.
The first report of chemical conversion of a ligand by an antibody appeared in 1980 (2), but the steroid ester hydrolysis by a rabbit polyclonal antiserum described in this report was stoichiometric rather than catalytic.
Massey et al, U.S. Pat. No. 4,888,281 were the first to report catalyzing a chemical reaction by means of an antibody elicited to a reactant, a reactant bound to a peptide or other carrier molecule, a reaction intermediate or analogs of the reactant, product or a reaction intermediate.
Subsequently, antibodies have been demonstrated to catalyze or facilitate chemical reactions, including acyl transfer (3-6), pericyclic (7-8) and redox reactions (9).
It is generally believed that reported antibodies (3-9) obtain their catalytic properties, like enzymes, from their ability to bind the transition state of the reactant better than its ground state.
Various analogs of the transition state of reactants have been used as antigens in the elicitation of immune responses (10).
The requirement for a hapten which antigenically mimics the transition state complicates efforts to obtain desirable catalytic antibodies. It would be advantageous if catalytic antibodies could be elicited to a ground state, as an antigen. Suckling et al. (11) have reported the use of a ground state antigen comprising a hapten related in structure to the substrate to elicit an antibody able to catalyze a Dieis-Alder reaction (the addition of acetoxybutadiene to N-substituted maleimides) and an antibody able to cleave beta lactam rings). Cleavage of peptide bonds by means of antibodies elicited to a selected peptide-metal complex has been demonstrated with the assistance of metal cofactors by Iverson et al. (12). Iverson et al. utilized a Co(III)triethylenetetramine (trien)-peptide hapten in order to elicit an antibody able to accept a metal complex with chemical reactivity into the binding pocket.
The discovery, isolation and characterization of naturally occurring autoantibodies, i.e., antibodies produced by an animal's immune system to the animal's own cellular component (self-antigen), as opposed to an antigen introduced by immunization, which enhance the rate of a chemical reaction, is disclosed in copending U.S. patent application Ser. No. 343,081, filed Apr. 4, 1989. These autoantibodies have been shown to enhance the rate of cleavage of one or more peptide bonds in vasoactive intestinal peptide (VIP). The natural occurrence of these catalytic autoantibodies in multiple humans suggests that there is a common, naturally occurring antigen capable of eliciting these autoantibodies. Classical catalytic antibody theory suggests that the naturally occurring autoantibody must be formed in response to a high energy transition state intermediate or in response to an unusually charged analog of the peptide. It would therefore not be expected that the antigen is the ground state of a peptide, or is a large precursor protein that is eventually digested to yield the peptide, for example, pro-VIP.
It is also known that antibody binding is energetically most favored by the presence of the entire H-chain and L-chain binding site (13). The V.sub.H fragments of anti-lysozyme antibodies bind the antigen with an affinity of only 10% of the intact antibody (14). L-chains are also likely to participate in antigen binding interactions, although most studies suggest that the contribution of L-chains is smaller than that of H-chains (15-17). It would not be expected that an antibody component smaller than an intact catalytic antibody would possess the favorable steric conformation provided by the intact catalytic antibody to permit the catalysis of a peptide bond without the assistance of a metal trien cofactor as taught by Iverson et al.
Improved antibodies and methods for selectively eliciting antibodies able to catalyze a chemical reaction of a peptide of interest are of singular interest for therapeutic products and other purposes.
The use of a ground state antigen would eliminate the necessity for preparing stable analogues of transition state intermediates or haptens complexed with metal co-factors so that catalytic antibodies specific for a polypeptide of interest may be elicited as needed.
There are obvious advantages that single chain proteins offer over multichain proteins (antibodies), both from the point of view of structure-function analysis as well as pharmacological and therapeutic stability. It would be advantageous if the binding and catalytic domains on an antibody were either the same or closely positioned to one another such that the benefits of catalytic activity could be achieved by a simple protein as opposed to a multichain antibody. Heretofore, the art has not demonstrated the capability of using such components of an antibody for catalytic purposes. Similar advantages are offered by dimers formed of the several combinations of light and heavy chains. Nor has the art demonstrated that an antibody light chain elicited by a ground state reactant will have catalytic activity.